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| Dissertation / PhD Thesis/Book | PreJuSER-13531 |
2001
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/19976
Report No.: Juel-3872
Abstract: Tat-dependent protein translocation was investigated in Escherichia coli and Z. mobilis with regard to species specific differences . E. coli is unable to export the authentic Tat-substrate glucose-fructose oxidoreductase (GFOR) of Zymombnas mobilis. The replacement of the Z. mobilis GFOR signal peptide by the E. coli Tat signal peptide of the Trimethylamin N-oxide (TMAO) reductase (TorA) leads to an efficient strictly Tat-dependent export of the mature GFOR in E. coli. This result shows that the GFOR signal sequence is not recognised by the E. coli Tat-apparatus, whereas the folded mature part of GFOR is compatible with Tatdependent translocation in E. coli. The tatABC operon of Z. mobilis was cloned . The single Tat proteins TatA, TatB and TatC of Z. mobilis are functional in E. coli and at least one of these proteins is involved in the specific recognition of the GFOR signal sequence (Tat signal peptide receptor) . Therefore, a specific recognition event between Tat substrate and Tat receptor takes place that goes beyond the recognition of the conserved general features found in all Tat signal peptides. The species specificity of signal sequence recognition in the Tat pathway is in marked contrast to the situation that is known to exist for the Sec pathway. In E. coli, only four different components of the Tat pathway are known so far. Therefore, a screening assay that is suitable for the identification of so far unknown tat-genes was established. In this assay, a TorA-MalE fusion protein was proved to be an ideal reporter protein for the Tat-pathway . Due to a strong Sec avoidance motive in the C-region of the TorA signal sequence, export of the fusion protein is strictly Tat-dependent. Export is essential for maltose metabolism in a maIE-negative E. coli strain and easy to detect on maltose-containing indicator plates. After mutagenesis of an E. coli Tat wildtype strain, mutants which are unable to metabolise maltose due to a mutation in genes of the Tat pathway or maltose metabolism, were selected from the indicator plates. The tat-mutants were identified by means of their inability to grow under certain anaerobic growth conditions. In a first test of the sceening assay, several mutants in the already known tatABC-operon were selectively isolated.
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